Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region

Abstract

Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.